Principal Investigator/Program Director: Evans, Christopher J., Ph.D., Component I Component II: Functional Imaging of Muand Delta Opioid Receptors In Vivo: Receptor Dynamics and Adaptation to Abused Opiates ABSTRACT: Opiates are important therapeutic compounds for treatment of pain, but they also have the potential to cause addiction. Studies have demonstrated specific roles for the different opioid receptors in the therapeutic and addictive effects of opiates Our recent studies using mice genetically lacking opioid receptors establish (i) the mu receptor as a key molecular trigger for both artificial and natural rewarding stimuli, and (ii)delta receptor activity as a possible factor in vulnerability to drug abuse and context-induced craving. The question of how these receptors operate within neural circuits and adapt to chronic opiates in vivo, is being widely studied at biochemical, electrophysiological and behavioral levels. However, this important issue remains poorly understood. A significant obstacle to the application of these techniques to the question of opiate receptor and addiction research has been the lack of reliable methods for directly visualizing opiate receptors in physiologically relevant models. The development of genetically encoded, fluorescent- labeled opioid receptors represents a straightforward path to overcome this obstacle. We have successfully developed a mouse that expresses the delta opioid receptor labeled with a fluorescent marker, Green Fluorescent Protein, and obtained fascinating imaging data. We have established the proof of principle that direct receptor visualization in mice in vivo is an achievable goal. We now plan to develop mice that express mu opioid receptors labeled with a different fluorescent marker, mCherry, as well as combinatorial mice expressing both types of labeled receptors. We will use cutting-edge in vitro and in vivo imaging techniques, in combination with advanced molecular techniques, to achieve the following Specific Aims: 1. To generate the MOR-mcherry knock-in mouse model and characterize receptor expression and distribution. 2. To study MOR-mcherry and DOR-eGFP neuroanatomy in mutant mouse lines by fluorescence microscopy. 3. To study MOR-mcherry and DOR-eGFP dynamics in response to acute opiates. 4. To study MOR-mcherry and DOR-eGFP dynamics in response to chronic opiates. Our project offers an entirely novel approach to achieve functional imaging of mu and delta opioid receptors in vivo. The ability to definitively identify neurons expressing each individual receptor subtype will represent a major advance in our ability to characterize the physiological responses of opioid receptor neurons under basal conditions, as well in response to acute and chronic opioids and other neurotransmitters. We will also be able to directly visualize the location of the expression of these receptors in the brain, their location in specific compartments of individual cells, and how the brain and cellular distribution of receptors changes under different physiological and pharmacological conditions. The combination of the latest imaging technologies and powerful new molecular tools for visualization of opioid receptors has the potential to provide important new insight into the cellular mechanisms by which opiates exert their therapeutic and addictive effects. Primary